Comparative MicroRNA Transcriptomics Of High-and Low-altitude Goats

High-altitude adaptation is a representative example of vertebrates’ adaptation to harsh and extreme environments,which serves as a natural model for studying the adaptive evolution of animals.In recent years,researchers have performed numerous studies on the mechanisms of high-altitude adaptation at levels of morphology,physiology,biochemistry,ethology,and genomics,but rarely focused on the roles miRNA plays in high-altitude adaptation,especially in agricultural animals.To reveal the molecular mechanisms of high-altitude adaptation in goat,we acquired small RNA transcriptomic data from highand low-altitude goat populations using small RNA sequencing methods,and integrated m RNA transcriptomic data from our previous study to investigate the mechanisms during high-altitude adaptation of goat.The main results are described as follows:(1)We performed constitution and sequencing of small RNA libraries on six hypoxia-sensitive tissues(heart,kidney,liver,lung,skeletal muscle,and spleen)in two goat populations from distinct altitudes(600 m and 3000 m),and annotated 1391 mature miRNAs and 909 pre-miRNAs.(2)The analysis of genomic sources indicated that most of pre-miRNAs(46.86%)were located in introns,followed by 37.95% in intergenic regions as the second largest category.Only a few miRNA precursors(0.32%)were located in exons.(3)The results of hierarchical clustering and principal components analysis(PCA)of global miRNA expression profiles showed that,samples were almost perfectly clustered by tissue and then by altitude.Among the six tissues,muscle and heart were more similar to each other than to other tissues.(4)Based on the comparison between tissue specificity of miRNAs and m RNAs,we found the miRNAs were more tissue-specific than protein-coding genes.Additionally,tissue-specific genes targeted by tissue-specific miRNAs were significantly enriched in functional categories specific to the corresponding tissue.(5)We identified 138 differentially expressed(DE)miRNAs in total.And muscle exhibited the highest number of DE miRNAs among the six tissues.Tissue specificity analysis based on DE miRNAs showed overall lower tissue specificity of DE miRNAs than that of non-differentially expressed miRNAs.(6)The overlap of DE miRNAs among tissues was low and most of the DE miRNAs(n = 91)underwent expression changes with no overlap among tissues.Compared with miRNAs that were differentially expressed in multiple tissues,miRNAs that were differentially expressed in one single tissue were more tissue-specific and their target genes were enriched in tissue-related functions;in contrast,the overlapping DE miRNAs with consistent regulation participated in more widespread processes in high-altitude adaptation.(7)By performing target prediction of DE miRNAs and functional enrichment analysis of target genes,we found that target genes were mainly enriched in functional categories including biological processes such as hypoxia response,apoptosis,DNA damage,angiogenesis,and protein-ubiquitination,as well as pathways such as HIF-1,p53,and insulin signaling pathways.(8)Integrating m RNA transcriptome data from our previous study,we found negative regulation between DE miRNAs and DE m RNAs in HIF-1,p53,and insulin signaling pathways,which were associated with processes such as angiogenesis,gluconeogenesis,glycogenesis,apoptosis,and DNA damage.(9)Functional experiments further confirmed miR-106a-5p to have a negative regulation effect on angiogenesis by directly targeting FLT-1.